Hemostasis is the mechanism by means of which living beings respond to a hemorrhage and involves the participation of two processes that become functional immediately after a lesion and remain active for a long period of time. The first of them is known as primary hemostasis and is characterized by the occurrence of vasoconstriction at the vascular lesion site and platelet aggregate formation. The second one is known as secondary hemostasis, being the phase in which the fibrin clot is formed due to the action of the different coagulation cascade proteolytic enzymes.
Several cofactors and proteolytic enzymes participate in the second phase of the blood coagulation process, all referred to as coagulation factors, and it consists of several phases ending with fibrin formation from fibrinogen hydrolysis due to the action of thrombin. Thrombin is previously formed by proteolytic hydrolysis of an apoenzyme, prothrombin. This proteolysis is carried out by the activated coagulation Factor X (FXa), which binds to the surface of the activated platelets and only in the presence of its cofactor, activated coagulation Factor V (FVa), and calcium ions, and is able to hydrolyze prothrombin. Coagulation Factor X (FX) activation can occur in two separate pathways, the intrinsic pathway and the extrinsic pathway.
The intrinsic pathway consists of a series of reactions in which each proenzyme is hydrolyzed, yielding its active protease form. In each step, the recently formed proteolytic enzyme will catalyze activation of the following proenzyme to successively yield the active form.
In the blood coagulation extrinsic pathway, the Tissue Factor (TF), exposed on adventitia cells at the lesion site, binds to circulating coagulation Factor VII/activated coagulation Factor VII (FVII/FVIa) to form the TF::FVIIa complex and, in the presence of calcium, to act as a substrate so that FX activation takes place. The extrinsic pathway is currently considered the most relevant pathway in blood coagulation, and it is accepted that in the event of a hemorrhage produced by a vascular lesion, coagulation is triggered due to extrinsic pathway activation involving the interaction of TF with its ligand, FVII/FVIIa.
TF consists of a protein component (previously referred to as tissue factor apoprotein-III) and a phospholipid. TF specifically binds to FVII/FVIIa and plays a relevant role in the blood coagulation extrinsic pathway. The physiological roles assigned to TF are well known; on the one hand, it is a receptor specific for FVIIa and, once the TF::FVIIa complex has been formed, it acts as a substrate so that FX activation takes place. In fact, after a vascular lesion, TF, which is normally sequestered on the surface of adventitia cells externally surrounding blood vessels, comes into contact and interacts with its ligand, FVII present in blood, to form the TF::FVII complex. Once this complex is formed, FVII autoactivation takes place, yielding its active form (FVIIa).
Glycosylation is an enzyme directed site specific process by which saccharides are added to lipids and proteins. It is believed that this process is involved in stability, folding, and transport; although no evidence of its real function has been described for TF.
It has been broadly accepted that TF is the main element responsible for the quickness with which coagulation is initiated. For coagulation to begin, it is absolutely necessary for FX to be activated and begin prothrombin hydrolysis. The source of this FXa has mainly been attributed to the interaction of FVIIa with its receptor, TF.
Purification of TF has been reported from various tissues such as: human brain, bovine brain; human placenta; ovine brain; and, lung. It is widely accepted that while there are differences in structure of TF protein between species there are no functional differences as measured by in vitro coagulation assays.
It is widely accepted that in order to demonstrate biological activity, TF must be associated with phospholipids in vitro. It has been shown that the removal of the phospholipid component of TF, for example by use of a phospholipase, results in a loss of its biological activity in vitro. Relipidation can restore in vitro TF activity.
While some quantities of “purified” TF protein have been available as obtained from various tissues, the low concentration of TF protein in blood and tissues and the high cost, both economic and of effort, of purifying the protein from tissues makes this a scarce material. Therefore, there is a need to look for an alternative source of TF protein, advantageously lipidated TF protein.
The TF protein has been expressed in various systems using the cloned human cDNA. Thus, over-expression of TF protein in E. coli has been reported (Paborsky et al., Biochemistry 28, 8072 (1989)). Further, U.S. Pat. No. 6,261,803 discloses a process for preparing functional recombinant TF in a prokaryotic host organism. High expression yield of the complete TF protein is achieved in E. coli. 
Although heterologous expression of proteins in E. coli presents some advantages, the expression of eukaryotic proteins in said bacteria is associated with a large number of problems, mainly when the protein to be expressed is a glycosylated eukaryotic protein, due to the lack in bacteria of their own glycosylation systems.
An alternative strategy consists, therefore, in expressing a mutated TF protein which lacks the transmembrane domain. This so-called “soluble” TF (or “truncated” TF) accumulates in the cytoplasm of the bacterial cells and can be expressed in relatively large quantities. However, in this system, the TF protein so expressed is usually present in E. coli in a quasicrystalline state in the form of so-called inclusion bodies. When this is the case, the inclusion bodies have to be solubilized by using very large quantities of chaotropic agents, and the proteins which have been monomerized in this way have then once again to be refolded, with a great deal of effort and usually with only a low yield, into an active, renatured confirmation. Further, in principle, the soluble TF is not suitable for use in prothrombin time reagents since it lacks the domain for the interaction with phospholipids.
Another approach for over-expressing TF protein is that of using a large number of known and successfully employed expression systems which encode products of gene fusions (e.g. with β-galactosidase, MalE, glutathione transferase, His-tag, etc.). However, these systems are not suitable for expressing biologically active TF. Although expression products can be detected and the level of expression can also be increased when said systems are used, the expression products so obtained are sometimes associated with a complete loss of function, which cannot be restored, either, even using elaborate renaturation methods.
The problems of over-expression of TF protein in E. coli can be circumvented by carrying out the expression of said protein in a eukaryotic system. Thus, expression in yeast cells, in insect cell cultures using baculovirus as a vector, or in cultured mammalian cells, e.g., hamster ovary cells, or in human cell lines, is, in principle, suitable. However, these systems suffer from crucial disadvantages, among others; recombinant protein yields are much lower when compared to recombinant E. coli production.
Yeast strains combine the advantages of the above distinct host systems. On one hand, they more closely mimic the native physiology of an eukaryotic protein than E. coli, and, on the other hand, they are ease of handling, ease of culturing, present much faster growth and much greater economy. Several factors though, affect the expression of proteins in yeast as well. These factors include, but are not confined to:                the choice of the gene regulatory sequences, such as promoters, that control the expression of an heterologous protein; the promoter sequences employed for controlling heterologous expression should typically be “strong”, i.e., they should effect very high expression of the protein, and suitably controllable, whereby the expression may at first be efficiently repressed until an optimum biomass of the culture is reached and then quickly switched on to effect protein expression; and        an efficient secretion of the expressed heterologous protein; secretion of the expressed protein (extracellular expression) is often preferred over intracellular expression as the latter would first entail breaking open the cell, thus disgorging the entire cellular contents, and then isolating the desired protein from the cesspool of cellular material and debris. Yet efficient secretion of a protein in turn depends on several factors including: (i) the choice of the signal sequences-peptide sequences which are usually the N-terminal regions of naturally secreted proteins, and which direct the protein into the cellular secretory pathway, and, (ii) the specific components of the secretory pathway that interact with signal sequences and effect the secretion of the attached protein.        
Stone M. J. et al (Biochem. J. (1995) 310, 605-614) describe the expression of the surface domain of TF (truncated TF) in Saccharomyces cerevisiae. For TF purification, cultures were loaded onto an immunoaffinity column conjugated with an anti-TF antibody and the protein is eluted and dialysed. This assay allowed access to milligram quantities of truncated TF.
Brucato C. L. et al. (Protein Expression and Purification 26 (2002), 386-393) describe expression of the mature full-length recombinant rabbit TF protein in Pichia pastoris. Purification of TF protein is carried out by immobilized metal-affinity chromatography.
There is so far no known process for preparing large quantities of biologically active, recombinant TF from yeast in high yield. Advantageously, said recombinant TF should be obtained at a high level of activity, preferably, at a level of activity suitable for therapeutical uses. Hence, it is an object of the present invention to generate useful quantities of lipidated TF protein using recombinant techniques. Advantageously, the recombinant TF protein should be useful for therapeutical applications.